Stromal interaction molecule 1 (STIM1) is involved in the regulation of mitochondrial shape and bioenergetics and plays a role in oxidative stress.

Abstract

Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Store-operated Ca(2+) entry over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and is mediated by the sensor STIM1 and the channel ORAI1. We compared cell death susceptibility to oxidative stress in STIM1 knock-out and ORAI1 knockdown mouse embryonic fibroblasts and in knock-out cells with reconstituted wild type and dominant active STIM1. We show that STIM1 and ORAI1 deficiency renders cells more susceptible to oxidative stress, which can be rescued by STIM1 and ORAI1 overexpression. STIM1 knock-out mitochondria are tubular, have a higher Ca(2+) concentration, and are metabolically more active, resulting in constitutive oxidative stress causing increased nuclear translocation of the antioxidant transcription factor NRF2 triggered by increased phosphorylation of the translation initiation factor eIF2α and the protein kinase-like endoplasmic reticulum kinase PERK. This leads to increased transcription of antioxidant genes and a high basal glutathione in STIM1 knock-out cells, which is, however, more rapidly expended upon additional stress, resulting in increased release and nuclear translocation of apoptosis-inducing factor with subsequent cell death. Our data suggest that store-operated Ca(2+) entry and STIM1 are involved in the regulation of mitochondrial shape and bioenergetics and play a role in oxidative stress.

Effects of STIM1 deficiency on the intracellular Ca2+ homeostasis. fura2 single-cell imaging of WT and STIM1 KO cells. After 1 min of base-line recording, 2 μm thapsigargin (A) or 5 μm ionomycin (C) was added as indicated, and the fura2 signal was recorded for an additional 4 min. The graphs show mean fura2 ratio of >150 cells for each condition. For all experiments, the area under the curve (A and C) and the base-line fura2 ratio (B) of the first minute of measurement was calculated and plotted as the means ± S.E. n.s., not significant; *, p < 0.05, Student's t test.

Effects of STIM1 rescue on the intracellular Ca2+ homeostasis.A, Fura2 single cell imaging of stably STIM1-EYFP or DA-STIM1-EYFP expressing STIM1 KO MEFs. After 1 min of base-line recording, 2 μm thapsigargin or 5 μm ionomycin was added as indicated, and the fura2 signal was recorded for additional 4 min. The graphs show mean fura2 ratio of >150 cells for each condition. For all experiments, the area under the curve (A) and the base-line fura2 ratio (B) of the first minute of measurement were calculated and plotted as the means ± S.E. n.s., not significant; *, p < 0.05, ANOVA with Tukey's post hoc test.

The up-regulated antioxidant response in STIM1 KO cells is mediated via NRF2 and phosphorylated eIF2α.A, translocation of NRF2 from the cytosol to the nucleus was analyzed by immunofluorescence in STIM1 WT and KO MEFs. NRF2 immunofluorescence intensity was quantified on a BD Pathway high content imaging system in the nucleus, which was identified by Hoechst staining, and the surrounding cytoplasm represented by a concentric ring around the nucleus. This method of segmentation is illustrated in the left panel. The ratio of nuclear to cytosolic NRF2 signal was calculated and normalized to WT signal. The graphs represent the means ± S.E. of 4468 WT and 3331 KO cells measured in three independent experiments each done in quintuplicates. B and C, immunoblot analysis of the phosphorylation state of eIF2α (B) or PERK (C). The blots were incubated with phosphorylation sensitive and insensitive antibodies against eIF2α (B) or phosphorylation-sensitive antibody against PERK and an antibody against actin as a loading control (C). Intensity values were calculated with the image analysis software ImageJ and normalized to WT control. The graphs represent the means ± S.E. of three independent experiments. n.s., not significant; *, p < 0.05, Student's t test.

Mitochondria of STIM1 KO cells are more densely packed and have a tubular shape.A, electron microscopic images of STIM1 WT and KO MEFs were captured on a Hitachi H 600 microscope, and several morphological parameters from 523 KO mitochondria distributed over 30 visual fields and 523 WT mitochondria in 32 visual fields were quantitated and expressed as the means ± S.E. B, length of the mitochondrial cut surface. C, number of direct contacts of mitochondria to ER membranes defined by a distance of less than 100 nm divided by the number of mitochondria. D, distance between mitochondria and the nearest ER membrane. E, typical pictures of the four categories of mitochondrial shape. Healthy cells are flat and outstretched, whereas apoptotic cells appear smaller and rounded. Tubular mitochondria show a long filamentous shape, whereas fragmented mitochondria appear small and rounded. F, quantitative analysis of mitochondrial shape of WT and STIM1 KO cells with or without glutamate treatment (2.5 mm overnight). 100 cells of each condition were analyzed with a 60× objective and categorized by their mitochondrial shape by a blinded investigator. The bar graphs represent the means ± S.E. of three independently performed experiments. n.s., not significant; *, p < 0.05, Student's t test. Tub., tubular mitochondria; Frag., fragmented mitochondria.